Antisense oligonucleotide modulation of raf gene expression

ABSTRACT

Oligonucleotides are provided which are targeted to nucleic acids encoding human raf and capable of inhibiting raf expression. The oligonucleotides may have chemical modifications at one or more positions and may be chimeric oligonucleotides. Methods of inhibiting the expression of human raf using oligonucleotides of the invention are also provided. The present invention further comprises methods of inhibiting hyperproliferation of cells and methods of treating or preventing conditions, including hyperproliferative conditions, associated with raf expression.

[0001] This application is a continuation of Ser. No. 09/506,073 filedFeb. 18, 2000, which is a continuation-in-part of Ser. No. 09/143,214filed Aug. 28, 1998 now issued as U.S. Pat. No. 6,090,626, which is acontinuation of Ser. No. 08/756,806 filed Nov. 26, 1996 now issued asU.S. Pat. No. 5,952,229, which is a continuation of PCT/US95/07111 filedMay 31, 1995 and Ser. No. 08/250,856 filed May 31, 1994 now issued asU.S. Pat. No. 5,563,255. This application is also a continuation-in-partof Ser. No. 08/888,982, filed Jul. 7, 1997 now issued as U.S. Pat. No.5,981,731 and corresponding PCT application PCT/US98/13961 filed Jul. 6,1998. Each of these applications is assigned to the assignee of thepresent invention.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for modulatingexpression of the raf gene, a naturally present cellular gene which hasbeen implicated in abnormal cell proliferation and tumor formation. Thisinvention is also directed to methods for inhibiting hyperproliferationof cells; these methods can be used diagnostically or therapeutically.Furthermore, this invention is directed to treatment of conditionsassociated with expression of the raf gene.

BACKGROUND OF THE INVENTION

[0003] Alterations in the cellular genes which directly or indirectlycontrol cell growth and differentiation are considered to be the maincause of cancer. The raf gene family includes three highly conservedgenes termed A-, B- and c-raf (also called raf-1). Raf genes encodeprotein kinases that are thought to play important regulatory roles insignal transduction processes that regulate cell proliferation.Expression of the c-raf protein is believed to play a role in abnormalcell proliferation since it has been reported that 60% of all lungcarcinoma cell lines express unusually high levels of c-raf mRNA andprotein. Rapp et al., The Oncogene Handbook, E. P. Reddy, A. M Skalkaand T. Curran, eds., Elsevier Science Publishers, New York, 1988, pp.213-253.

[0004] Oligonucleotides have been employed as therapeutic moieties inthe treatment of disease states in animals and man. For example, workersin the field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and clinicaltrials of several antisense oligonucleotide drugs, targeted both toviral and cellular gene products, are presently underway. Thephosphorothioate oligonucleotide drug, Vitravene™ (ISIS 2922), has beenapproved by the FDA for treatment of cytomegalovirus retinitis in AIDSpatients. It is thus established that oligonucleotides can be usefultherapeutic instrumentalities and can be configured to be useful intreatment regimes for treatment of cells and animal subjects, especiallyhumans.

[0005] Antisense oligonucleotide inhibition of gene expression hasproven to be a useful tool in understanding the roles of raf genes. Anantisense oligonucleotide complementary to the first six codons of humanc-raf has been used to demonstrate that the mitogenic response of Tcells to interleukin-2 (IL-2) requires c-raf. Cells treated with theoligonucleotide showed a near-total loss of c-raf protein and asubstantial reduction in proliferative response to IL-2. Riedel et al.,Eur. J. Immunol. 1993, 23, 3146-3150. Rapp et al. have disclosedexpression vectors containing a raf gene in an antisense orientationdownstream of a promoter, and methods of inhibiting raf expression byexpressing an antisense Raf gene or a mutated Raf gene in a cell. WOapplication 93/04170. An antisense oligodeoxyribonucleotidecomplementary to codons 1-6 of murine c-Raf has been used to abolishinsulin stimulation of DNA synthesis in the rat hepatoma cell lineH4IIE. Tornkvist et al., J. Biol. Chem. 1994, 269, 13919-13921. WOApplication 93/06248 discloses methods for identifying an individual atincreased risk of developing cancer and for determining a prognosis andproper treatment of patients afflicted with cancer comprising amplifyinga region of the c-raf gene and analyzing it for evidence of mutation.

[0006] Denner et al. disclose antisense polynucleotides hybridizing tothe gene for raf, and processes using them. WO 94/15645.Oligonucleotides hybridizing to human and rat raf sequences aredisclosed.

[0007] Iversen et al. disclose heterotypic antisense oligonucleotidescomplementary to raf which are able to kill ras-activated cancer cells,and methods of killing raf-activated cancer cells. Numerousoligonucleotide sequences are disclosed, none of which are actuallyantisense oligonucleotide sequences.

[0008] There remains a long-felt need for improved compositions andmethods for inhibiting raf gene expression.

SUMMARY OF THE INVENTION

[0009] The present invention provides oligonucleotides which aretargeted to nucleic acids encoding human raf and are capable ofinhibiting raf expression. The present invention also provides chimericoligonucleotides targeted to nucleic acids encoding human raf. Theoligonucleotides of the invention are believed to be useful bothdiagnostically and therapeutically, and are believed to be particularlyuseful in the methods of the present invention.

[0010] The present invention also comprises methods of inhibiting theexpression of human raf, particularly the abnormal expression of raf.These methods are believed to be useful both therapeutically anddiagnostically as a consequence of the association between rafexpression and hyperproliferation. These methods are also useful astools, for example for detecting and determining the role of rafexpression in various cell functions and physiological processes andconditions and for diagnosing conditions associated with raf expression.

[0011] The present invention also comprises methods of inhibitinghyperproliferation of cells using oligonucleotides of the invention.These methods are believed to be useful, for example in diagnosingraf-associated cell hyperproliferation. These methods employ theoligonucleotides of the invention. These methods are believed to beuseful both therapeutically and as clinical research and diagnostictools.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Malignant tumors develop through a series of stepwise,progressive changes that lead to the loss of growth controlcharacteristic of cancer cells, i.e., continuous unregulatedproliferation, the ability to invade surrounding tissues, and theability to metastasize to different organ sites. Carefully controlled invitro studies have helped define the factors that characterize thegrowth of normal and neoplastic cells and have led to the identificationof specific proteins that control cell growth and differentiation. Theraf genes are members of a gene family which encode related proteinstermed A-, B- and c-raf. Raf genes code for highly conservedserine-threonine-specific protein kinases. These enzymes aredifferentially expressed; c-raf, the most thoroughly characterized, isexpressed in all organs and in all cell lines that have been examined.A- and B-raf are expressed in urogenital and brain tissues,respectively. c-raf protein kinase activity and subcellular distributionare regulated by mitogens via phosphorylation. Various growth factors,including epidermal growth factor, acidic fibroblast growth factor,platelet-derived growth factor, insulin, granulocyte-macrophagecolony-stimulating factor, interleukin-2, interleukin-3 anderythropoietin, have been shown to induce phosphorylation of c-raf.Thus, c-raf is believed to play a fundamental role in the normalcellular signal transduction pathway, coupling a multitude of growthfactors to their net effect, cellular proliferation.

[0013] Certain abnormal proliferative conditions are believed to beassociated with raf expression and are, therefore, believed to beresponsive to inhibition of raf expression. Abnormally high levels ofexpression of the raf protein are also implicated in transformation andabnormal cell proliferation. These abnormal proliferative conditions arealso believed to be responsive to inhibition of raf expression. Examplesof abnormal proliferative conditions are hyperproliferative disorderssuch as cancers, tumors, hyperplasias, pulmonary fibrosis, angiogenesis,psoriasis, atherosclerosis and smooth muscle cell proliferation in theblood vessels, such as stenosis or restenosis following angioplasty. Thecellular signaling pathway of which raf is a part has also beenimplicated in inflammatory disorders characterized by T-cellproliferation (T-cell activation and growth), such as tissue graftrejection, endotoxin shock, and glomerular nephritis, for example.

[0014] It has now been found that elimination or reduction of raf geneexpression may halt or reverse abnormal cell proliferation. This hasbeen found even in when levels of raf expression are not abnormallyhigh. There is a great desire to provide compositions of matter whichcan modulate the expression of the raf gene. It is greatly desired toprovide methods of detection of the raf gene in cells, tissues andanimals. It is also desired to provide methods of diagnosis andtreatment of abnormal proliferative conditions associated with abnormalraf gene expression. In addition, kits and reagents for detection andstudy of the raf gene are desired. “Abnormal” raf gene expression isdefined herein as abnormally high levels of expression of the rafprotein, or any level of raf expression in an abnormal proliferativecondition or state.

[0015] The present invention employs oligonucleotides targeted tonucleic acids encoding raf. This relationship between an oligonucleotideand its complementary nucleic acid target to which it hybridizes iscommonly referred to as “antisense”. “Targeting” an oligonucleotide to achosen nucleic acid target, in the context of this invention, is amultistep process. The process usually begins with identifying a nucleicacid sequence whose function is to be modulated. This may be, asexamples, a cellular gene (or mRNA made from the gene) whose expressionis associated with a particular disease state, or a foreign nucleic acidfrom an infectious agent. In the present invention, the target is anucleic acid encoding raf; in other words, the raf gene or mRNAexpressed from the raf gene. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe oligonucleotide interaction to occur such that the desiredeffect—modulation of gene expression—will result. Once the target siteor sites have been identified, oligonucleotides are chosen which aresufficiently complementary to the target, i.e., hybridize sufficientlywell and with sufficient specificity, to give the desired modulation.

[0016] In the context of this invention “modulation” means eitherinhibition or stimulation. Inhibition of raf gene expression ispresently the preferred form of modulation. This modulation can bemeasured in ways which are routine in the art, for example by Northernblot assay of mRNA expression or Western blot assay of proteinexpression as taught in the examples of the instant application. Effectson cell proliferation or tumor cell growth can also be measured, astaught in the examples of the instant application. “Hybridization”, inthe context of this invention, means hydrogen bonding, also known asWatson-Crick base pairing, between complementary bases, usually onopposite nucleic acid strands or two regions of a nucleic acid strand.Guanine and cytosine are examples of complementary bases which are knownto form three hydrogen bonds between them. Adenine and thymine areexamples of complementary bases which form two hydrogen bonds betweenthem. “Specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when binding of the oligonucleotide to the targetinterferes with the normal function of the target molecule to cause aloss of utility, and there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired, i.e.,under physiological conditions in the case of in vivo assays ortherapeutic treatment or, in the case of in vitro assays, underconditions in which the assays are conducted.

[0017] In preferred embodiments of this invention, oligonucleotides areprovided which are targeted to mRNA encoding c-raf, A-raf and B-raf. Inaccordance with this invention, persons of ordinary skill in the artwill understand that mRNA includes not only the coding region whichcarries the information to encode a protein using the three lettergenetic code, but also associated ribonucleotides which form a regionknown to such persons as the 5′-untranslated region, the 3′-untranslatedregion, the 5′ cap region, intron regions and intron/exon or splicejunction ribonucleotides. Thus, oligonucleotides may be formulated inaccordance with this invention which are targeted wholly or in part tothese associated ribonucleotides as well as to the codingribonucleotides. In preferred embodiments, the oligonucleotide istargeted to a translation initiation site (AUG codon) or sequences inthe 5′- or 3′-untranslated region of the human c-raf mRNA. The functionsof messenger RNA to be interfered with include all vital functions suchas translocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing or maturation of the RNAand possibly even independent catalytic activity which may be engaged inby the RNA. The overall effect of such interference with the RNAfunction is to cause interference with raf protein expression.

[0018] The present invention provides oligonucleotides for modulation ofraf gene expression. Such oligonucleotides are targeted to nucleic acidsencoding raf. oligonucleotides and methods for modulation of c-raf,A-raf and B-raf are presently preferred; however, compositions andmethods for modulating expression of other forms of raf are alsobelieved to have utility and are comprehended by this invention. Ashereinbefore defined, “modulation” means either inhibition orstimulation. Inhibition of raf gene expression is presently thepreferred form of modulation.

[0019] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar(backbone) linkages. The term “oligonucleotide” also includes oligomerscomprising non-naturally occurring monomers, or portions thereof, whichfunction similarly. Such modified or substituted oligonucleotides areoften preferred over native forms because of properties such as, forexample, enhanced cellular uptake and increased stability in thepresence of nucleases.

[0020] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. Certain preferredoligonucleotides of this invention are chimeric oligonucleotides.“Chimeric oligonucleotides” or “chimeras”, in the context of thisinvention, are oligonucleotides which contain two or more chemicallydistinct regions, each made up of at least one nucleotide. Theseoligonucleotides typically contain at least one region of modifiednucleotides that confers one or more beneficial properties (such as, forexample, increased nuclease resistance, increased uptake into cells,increased binding affinity for the RNA target) and a region that is asubstrate for RNase H cleavage. In one preferred embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in this casea nucleic acid encoding raf) is routinely determined by measuring the Tmof an oligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target. In a more preferred embodiment, theregion of the oligonucleotide which is modified to increase raf mRNAbinding affinity comprises at least one nucleotide modified at the 2′position of the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkylor 2′-fluoro-modified nucleotide. Such modifications are routinelyincorporated into oligonucleotides and these oligonucleotides have beenshown to have a higher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target. The effect of suchincreased affinity is to greatly enhance antisense oligonucleotideinhibition of raf gene expression. RNAse H is a cellular endonucleasethat cleaves the RNA strand of RNA:DNA duplexes; activation of thisenzyme therefore results in cleavage of the RNA target, and thus cangreatly enhance the efficiency of antisense inhibition. Cleavage of theRNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance.

[0021] The oligonucleotides in accordance with this invention preferablyare from about 8 to about 50 nucleotides in length. In the context ofthis invention it is understood that this encompasses non-naturallyoccurring oligomers as hereinbefore described, having 8 to 50 monomers.Particularly preferred are antisense oligonucleotides comprising fromabout 8 to about 30 nucleobases (i.e. from about 8 to about 30 linkednucleosides).

[0022] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0023] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0024] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acidforms are also included.

[0025] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; and 5,625,050.

[0026] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

[0027] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439.

[0028] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al. (Science, 1991, 254,1497-1500).

[0029] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.5,034,506.

[0030] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl,O-alkyl-O-alkyl, O—, S—, or N-alkenyl, or O—, S— or N-alkynyl, whereinthe alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ toC₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. Other preferred oligonucleotides compriseone of the following at the 2′ position: C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Furtherpreferred modifications include 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and2′-dimethylaminoethoxyethoxy (2′-DMAEOE) as described in exampleshereinbelow.

[0031] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 35 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920.

[0032] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C orm5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, those disclosed by Englisch et al. (Angewandte Chemie,International Edition 1991, 30, 613-722), and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993,pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications 1993, CRC Press, Boca Raton, pages 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0033] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941.

[0034] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993,3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanovet al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937)

[0035] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0036] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling VA) to synthesize fluorescently labeled, biotinylatedor other modified oligonucleotides such as cholesterol-modifiedoligonucleotides.

[0037] It has now been found that certain oligonucleotides targeted toportions of the c-raf mRNA are particularly useful for inhibiting rafexpression and for interfering with cell hyperproliferation. Methods forinhibiting c-raf expression using antisense oligonucleotides are,likewise, useful for interfering with cell hyperproliferation. In themethods of the invention, tissues or cells are contacted witholigonucleotides. In the context of this invention, to “contact” tissuesor cells with an oligonucleotide or oligonucleotides means to add theoligonucleotide(s), usually in a liquid carrier, to a cell suspension ortissue sample, either in vitro or ex vivo, or to administer theoligonucleotide(s) to cells or tissues within an animal.

[0038] For therapeutics, methods of inhibiting hyperproliferation ofcells and methods of treating abnormal proliferative conditions areprovided. The formulation of therapeutic compositions and theirsubsequent administration is believed to be within the skill in the art.In general, for therapeutics, a patient suspected of needing suchtherapy is given an oligonucleotide in accordance with the invention,commonly in a pharmaceutically acceptable carrier, in amounts and forperiods which will vary depending upon the nature of the particulardisease, its severity and the patient's overall condition. Thepharmaceutical compositions of this invention may be administered in anumber of ways depending upon whether local or systemic treatment isdesired, and upon the area to be treated. Administration may be topical(including ophthalmic, vaginal, rectal, intranasal), oral, orparenteral, for example by intravenous drip, intravenous injection orsubcutaneous, intraperitoneal, intraocular, intravitreal orintramuscular injection.

[0039] Formulations for topical administration may include ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable. Coatedcondoms, gloves and the like may also be useful.

[0040] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0041] Formulations for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives.

[0042] In addition to such pharmaceutical carriers, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is Lipofectin (BRL, BethesdaMd.).

[0043] Compositions for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. In some cases it may be more effective to treat apatient with an oligonucleotide of the invention in conjunction withother traditional therapeutic modalities in order to increase theefficacy of a treatment regimen. In the context of the invention, theterm “treatment regimen” is meant to encompass therapeutic, palliativeand prophylactic modalities. For example, a patient may be treated withconventional chemotherapeutic agents, particularly those used for tumorand cancer treatment. Examples of such chemotherapeutic agents includebut are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, teniposide,cisplatin, carboplatin, topotecan, irinotecan, gemcitabine anddiethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosisand Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway,N.J. When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Other drugssuch as leucovorin, which is a form of folic acid used as a “rescue”after high doses of methotrexate or other folic acid agonists, may alsobe administered. In some embodiments, 5-FU and leucovorin are given incombination as an IV bolus with the compounds of the invention beingprovided as an IV infusion.

[0044] Dosing is dependent on severity and responsiveness of thecondition to be treated, with course of treatment lasting from severaldays to several months or until a cure is effected or a diminution ofdisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body. Persons of ordinaryskill can easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual oligonucleotides, and can generally be calculatedbased on EC50's in in vitro and in vivo animal studies. For example,given the molecular weight of compound (derived from oligonucleotidesequence and chemical structure) and an effective dose such as an IC50,for example (derived experimentally), a dose in mg/kg is routinelycalculated.

[0045] The present invention is also suitable for diagnosing abnormalproliferative states in tissue or other samples from patients suspectedof having a hyperproliferative disease such as cancer, psoriasis orblood vessel restenosis or atherosclerosis. The ability of theoligonucleotides of the present invention to inhibit cell proliferationmay be employed to diagnose such states. A number of assays may beformulated employing the present invention, which assays will commonlycomprise contacting a tissue sample with an oligonucleotide of theinvention under conditions selected to permit detection and, usually,quantitation of such inhibition. Similarly, the present invention can beused to distinguish raf-associated tumors from tumors having otheretiologies, in order that an efficacious treatment regime can bedesigned.

[0046] The oligonucleotides of this invention may also be used forresearch purposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

[0047] The oligonucleotides of the invention are also useful fordetection and diagnosis of raf expression. For example, radiolabeledoligonucleotides can be prepared by ³²P labeling at the 5′ end withpolynucleotide kinase. Sambrook et al., Molecular Cloning. A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, p. 10.59.Radiolabeled oligonucleotides are then contacted with tissue or cellsamples suspected of raf expression and the sample is washed to removeunbound oligonucleotide. Radioactivity remaining in the sample indicatesbound oligonucleotide (which in turn indicates the presence of raf) andcan be quantitated using a scintillation counter or other routine means.Radiolabeled oligo can also be used to perform autoradiography oftissues to determine the localization, distribution and quantitation ofraf expression for research, diagnostic or therapeutic purposes. In suchstudies, tissue sections are treated with radiolabeled oligonucleotideand washed as described above, then exposed to photographic emulsionaccording to routine autoradiography procedures. The emulsion, whendeveloped, yields an image of silver grains over the regions expressingraf. Quantitation of the silver grains permits raf expression to bedetected.

[0048] Analogous assays for fluorescent detection of raf expression canbe developed using oligonucleotides of the invention which areconjugated with fluorescein or other fluorescent tag instead ofradiolabeling. Such conjugations are routinely accomplished during solidphase synthesis using fluorescently labeled amidites or CPG (e.g.,fluorescein-labeled amidites and CPG available from Glen Research,Sterling Va. See 1993 Catalog of Products for DNA Research, GlenResearch, Sterling Va., p. 21).

[0049] Each of these assay formats is known in the art. One of skillcould easily adapt these known assays for detection of raf expression inaccordance with the teachings of the invention providing a novel anduseful means to detect raf expression.

[0050] Oligonucleotide Inhibition of C-raf Expression

[0051] The oligonucleotides shown in Table 1 were designed using theGenbank c-raf sequence HSRAFR (Genbank accession no. x03484; SEQ ID NO:64), synthesized and tested for inhibition of c-raf mRNA expression inT24 bladder carcinoma cells using a Northern blot assay. All areoligodeoxynucleotides with phosphorothioate backbones. TABLE 1 Humanc-raf Kinase Antisense Oligonucleotides SEQ ID Isis # Sequence (5′ → 3′)Site NO: 5000 TGAAGGTGAGCTGGAGCCAT Coding 1 5074 GCTCCATTGATGCAGCTTAAAUG 2 5075 CCCTGTATGTGCTCCATTGA AUG 3 5076 GGTGCAAAGTCAACTAGAAG STOP 45097 ATTCTTAAACCTGAGGGAGC 5′UTR 5 5098 GATGCAGCTTAAACAATTCT 5′UTR 6 5131CAGCACTGCAAATGGCTTCC 3′UTR 7 5132 TCCCGCCTGTGACATGCATT 3′UTR 8 5133GCCGAGTGCCTTGCCTGGAA 3′UTR 9 5148 AGAGATGCAGCTGGAGCCAT Coding 10 5149AGGTGAAGGCCTGGAGCCAT Coding 11 6721 GTCTGGCGCTGCACCACTCT 3′UTR 12 6722CTGATTTCCAAAATCCCATG 3′UTR 13 6731 CTGGGCTGTTTGGTGCCTTA 3′UTR 14 6723TCAGGGCTGGACTGCCTGCT 3′UTR 15 7825 GGTGAGGGAGCGGGAGGCGG 5′UTR 16 7826CGCTCCTCCTCCCCGCGGCG 5′UTR 17 7827 TTCGGCGGCAGCTTCTCGCC 5′UTR 18 7828GCCGCCCCAACGTCCTGTCG 5′UTR 19 7848 TCCTCCTCCCCGCGGCGGGT 5′UTR 20 7849CTCGCCCGCTCCTCCTCCCC 5′UTR 21 7847 CTGGCTTCTCCTCCTCCCCT 3′UTR 22 8034CGGGAGGCGGTCACATTCGG 5′UTR 23 8094 TCTGGCGCTGCACCACTCTC 3′UTR 24

[0052] In a first round screen of oligonucleotides at concentrations of100 nM or 200 nM, oligonucleotides 5074, 5075, 5132, 8034, 7826, 7827and 7828 showed at least 50% inhibition of c-raf mRNA and theseoligonucleotides are therefore preferred. Oligonucleotides 5132 and 7826(SEQ ID NO: 8 and SEQ ID NO: 17) showed greater than about 90%inhibition and are more preferred. In additional assays,oligonucleotides 6721, 7848, 7847 and 8094 decreased c-raf mRNA levelsby greater than 50%. These oligonucleotides are also preferred. Ofthese, 7847 (SEQ ID NO: 22) showed greater than about 90% inhibition ofc-raf mRNA and is more preferred.

[0053] Specificity of ISIS 5132 for raf

[0054] Specificity of ISIS 5132 for raf mRNA was demonstrated by aNorthern blot assay in which this oligonucleotide was tested for theability to inhibit Ha-ras mRNA as well as c-raf mRNA in T24 cells.Ha-ras is a cellular oncogene which is implicated in transformation andtumorigenesis. ISIS 5132 was shown to abolish c-raf mRNA almostcompletely with no effect on Ha-ras mRNA levels.

[0055] 2′-modified Oligonucleotides

[0056] Certain of these oligonucleotides were synthesized with eitherphosphodiester (P═O) or phosphorothioate (P═S) backbones and were alsouniformly substituted at the 2′ position of the sugar with either a2′-O-methyl, 2′-O-propyl, or 2′-fluoro group. Oligonucleotides are shownin Table 2. TABLE 2 Uniformly 2′ Sugar-modified c-raf OligonucleotidesSEQ ID ISIS # Sequence Site Modif NO. 6712 TCCCGCCTGTGACATGCATT 3′UTROMe/P = S 8 8033 CGGGAGGCGGTCACATTCGG 5′UTR OMe/P = S 23 7829GGTGAGGGAGCGGGAGGCGG 5′UTR OMe/P = S 16 7830 CGCTCCTCCTCCCCGCGGCG 5′UTROMe/P = S 17 7831 TTCGGCGGCAGCTTCTCGCC 5′UTR OMe/P = S 18 7832GCCGCCCCAACGTCCTGTCG 5′UTR OMe/P = S 19 7833 ATTCTTAAACCTGAGGGAGC 5′UTROMe/P = S 5 7834 GATGCAGCTTAAACAATTCT 5′UTR OMe/P = S 6 7835GCTCCATTGATGCAGCTTAA AUG OMe/P = S 2 7836 CCCTGTATGTGCTCCATTGA AUGOMe/P = S 3 8035 CGGGAGGCGGTCACATTCGG 5′UTR OPr/P = O 23 7837GGTGAGGGAGCGGGAGGCGG 5′UTR OPr/P = O 16 7838 CGCTCCTCCTCCCCGCGGCG 5′UTROPr/P = O 17 7839 TTCGGCGGCAGCTTCTCGCC 5′UTR OPr/P = O 18 7840GCCGCCCCAACGTCCTGTCG 5′UTR OPr/P = O 19 7841 ATTCTTAAACCTGAGGGAGC 5′UTROPr/P = O 5 7842 GATGCAGCTTAAACAATTCT 5′UTR OPr/P = O 6 7843GCTCCATTGATGCAGCTTAA AUG OPr/P = O 2 7844 CCCTGTATGTGCTCCATTCA AUGOPr/P = O 3 9355 CGGGAGGCGGTCACATTCGG 5′UTR 2′F/P = S 23

[0057] Oligonucleotides from Table 2 having uniform 2′O-methylmodifications and a phosphorothioate backbone were tested for ability toinhibit c-raf protein expression in T24 cells as determined by Westernblot assay. Oligonucleotides 8033, 7834 and 7835 showed the greatestinhibition and are preferred. Of these, 8033 and 7834 are morepreferred.

[0058] Chimeric Oligonucleotides

[0059] Chimeric oligonucleotides having SEQ ID NO: 8 were prepared.These oligonucleotides had central “gap” regions of 6, 8, or 10deoxynucleotides flanked by two regions of 2′-O-methyl modifiednucleotides. Backbones were uniformly phosphorothioate. In Northern blotanalysis, all three of these oligonucleotides (ISIS 6720, 6-deoxy gap;ISIS 6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap) showed greater than 70%inhibition of c-raf mRNA expression in T24 cells. These oligonucleotidesare preferred. The 8-deoxy gap compound (6717) showed greater than 90%inhibition and is more preferred.

[0060] Additional chimeric oligonucleotides were synthesized having oneor more regions of 2′-O-methyl modification and uniform phosphorothioatebackbones. These are shown in Table 3. All are phosphorothioates; boldregions indicate 2′-O-methyl modified regions. TABLE 3 Chimeric2′-O-methyl P = S c-raf oligonucleotides Target SEQ ID Isis # Sequencesite NO: 7848 TCCTCCTCCCCGCGGCGGGT 5′UTR 20 7352 TCCTCCTCCCCGCGGCGGGT5′UTR 20 7849 CTCGCCCGCTCCTCCTCCCC 5′UTR 21 7851 CTCGCCCGCTCCTCCTCCCC5′UTR 21 7856 TTCTCGCCCGCTCCTCCTCC 5′UTR 25 7855 TTCTCGCCCGCTCCTCCTCC5′UTR 25 7854 TTCTCCTCCTCCCCTGGCAG 3′UTR 26 7347 CTGGCTTCTCCTCCTCCCCT3′UTR 22 7850 CTGGCTTCTCCTCCTCCCCT 3′UTR 22 7853 CCTGCTGGCTTCTCCTCCTC3′UTR 27

[0061] When tested for their ability to inhibit c-raf mRNA by Northernblot analysis, ISIS 7848, 7849, 7851, 7856, 7855, 7854, 7847, and 7853gave better than 70% inhibition and are therefore preferred. Of these,7851, 7855, 7847 and 7853 gave greater than 90% inhibition and are morepreferred.

[0062] Additional chimeric oligonucleotides with various 2′modifications were prepared and tested. These are shown in Table 4. Allare phosphorothioates; bold regions indicate 2′-modified regions. TABLE4 Chimeric 2′-modified P = S c-raf oligonucleotides Target Modifi- SEQID Isis # SEQUENCE site cation NO: 6720 TCCCGCCTGTGACATGCATT 3′UTR2′-O-Me 8 6717 TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Me 8 6729TCCCGCCTGTGACATGCATT 3′UTR 2′-O-Me 8 8097 TCTGGCGCTGCACCACTCTC 3′UTR2′-O-Me 24 9270 TCCCGCCTGTGACATGCATT 3′UTR  2′-O-Pro 8 9058TCCCGCCTGTGACATGCATT 3′UTR 2′-F  8 9057 TCTGGCGCTGCACCACTCTC 3′UTR 2′-F 24

[0063] Of these, oligonucleotides 6720, 6717, 6729, 9720 and 9058 arepreferred. Oligonucleotides 6717, 6729, 9720 and 9058 are morepreferred.

[0064] Two chimeric oligonucleotides with 2′-O-propyl sugarmodifications and chimeric P═O/P═S backbones were also synthesized.These are shown in Table 5, in which italic regions indicate regionswhich are both 2′-modified and have phosphodiester backbones. TABLE 5Chimeric 2′-modified P = S/P = O c-raf oligonucleotides Target Modifi-SEQ ID Isis # Sequence site cation NO: 9271 TCCCGCCTGTGACATGCATT 3′UTR2′-O-Pro 8 8096 TCTGGCGCTGCACCACTCTC 3′UTR 2′-O-Pro 24

[0065] Inhibition of Cancer Cell Proliferation

[0066] The phosphorothioate oligonucleotide ISIS 5132 was shown toinhibit T24 bladder cancer cell proliferation. Cells were treated withvarious concentrations of oligonucleotide in conjunction with lipofectin(cationic lipid which increases uptake of oligonucleotide). Adose-dependent inhibition of cell proliferation was demonstrated, asindicated in Table 6, in which “None” indicates untreated control (nooligonucleotide) and “Control” indicates treatment with negative controloligonucleotide. Results are shown as percent inhibition compared tountreated control. TABLE 6 Inhibition of T24 Cell Proliferation by ISIS5132 Oligo conc. None Control 5132  50 nM 0 +9% 23% 100 nM 0 +4% 24% 250nM 0 10% 74% 500 nM 0 18% 82%

[0067] Effect of ISIS 5132 on T24 Human Bladder Carcinoma Tumors

[0068] Subcutaneous human T24 bladder carcinoma xenografts in nude micewere established and treated with ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide administered intraperitoneally threetimes weekly at a dosage of 25 mg/kg. In this preliminary study, ISIS5132 inhibited tumor growth after eleven days by 35% compared tocontrols. Oligonucleotide-treated tumors remained smaller than controltumors throughout the course of the study.

[0069] Antisense Oligonucleotides Targeted to A-raf

[0070] It is believed that certain oligonucleotides targeted to portionsof the A-raf mRNA and which inhibit A-raf expression will be useful forinterfering with cell hyperproliferation. Methods for inhibiting A-rafexpression using such antisense oligonucleotides are, likewise, believedto be useful for interfering with cell hyperproliferation.

[0071] The phosphorothioate deoxyoligonucleotides shown in Table 7 weredesigned and synthesized using the Genbank A-raf sequence HUMARAFIR(Genbank listing x04790; SEQ ID NO: 65). TABLE 7 OligonucleotidesTargeted to Human A-raf Target +HC,27, SEQ ID Isis # Sequence site NO:9060 GTC AAG ATG GGC TGA GGT CC 5′ UTR 23 9061 CCA TCC CGG ACA GTC ACCAC Coding 29 9062 ATG AGC TCC TCG CCA TCC AG Coding 30 9063 AAT GCT GGTGGA ACT TGT AG Coding 31 9064 CCG GTA CCC CAG GTT CTT CA Coding 32 9065CTG GGC AGT CTG CCG GGC CA Coding 33 9066 CAC CTC AGC TGC CAT CCA CACoding 34 9067 GAG ATT TTG CTG AGG TCC GG Coding 35 9068 GCA CTC CGC TCAATC TTG GG Coding 36 9069 CTA AGG CAC AAG GCG CCC TC Stop 37 9070 ACGAAC ATT GAT TGG CTG GT 3′ UTR 38 9071 GTA TCC CCA AAG CCA AGA GG 3′ UTR39 10228 CAT CAG CGC AGA GAC GAA CA 3′ UTR 40

[0072] Oligonucleotides ISIS 9061, ISIS 9069 and ISIS 10228 wereevaluated by Northern blot analysis for their effects on A-raf mRNAlevels in A549, T24 and NHDF cells. All three oligonucleotides decreasedA-raf RNA levels in a dose-dependent manner in all three cell types,with inhibition of greater than 50% at a 500 nM dose in all cell types.The greatest inhibition (88%) was achieved with ISIS 9061 and 9069 inT24 cells. These three oligonucleotides (ISIS 9061, 9069 and 10228) arepreferred, with ISIS 9069 and 9061 being more preferred.

[0073] Identification of Oligonucleotides Targeted to Rat and Mousec-raf

[0074] Many conditions which are believed to be mediated by raf kinaseare not amenable to study in humans. For example, tissue graft rejectionis a condition which is likely to be ameliorated by interference withraf expression; but, clearly, this must be evaluated in animals ratherthan human transplant patients. Another such example is restenosis.These conditions can be tested in animal models, however, such as therat and mouse models used here.

[0075] Oligonucleotide sequences for inhibiting c-raf expression in ratand mouse cells were identified. Rat and mouse c-raf genes have regionsof high homology; a series of oligonucleotides which target both rat andmouse c-raf mRNA sequence were designed and synthesized, usinginformation gained from evaluation of oligonucleotides targeted to humanc-raf. These oligonucleotides were screened for activity in mouse bENDcells and rat A-10 cells using Northern blot assays. Theoligonucleotides (all phosphorothioates) are shown in Table 8. TABLE 8Oligonucleotides targeted to mouse and rat c-raf Target SEQ ID ISIS #site Sequence NO: 10705 Coding GGAACATCTGGAATTTGGTC 41 10706 CodingGATTCACTGTGACTTCGAAT 42 10707 3′UTR GCTTCCATTTCCAGGGCAGG 43 10708 3′UTRAAGAAGGCAATATGAAGTTA 44 10709 3′UTR GTGGTGCCTGCTGACTCTTC 45 10710 3′UTRCTGGTGGCCTAAGAACAGCT 46 10711 AUG GTATGTGCTCCATTGATGCA 47 10712 AUGTCCCTGTATGTGCTCCATTG 48 11060 5′UTR ATACTTATACCTGAGGGAGC 49 11061 5′UTRATGCATTCTGCCCCCAAGGA 50 11062 3′UTR GACTTGTATACCTCTGGAGC 51 11063 3′UTRACTGGCACTGCACCACTGTC 52 11064 3′UTR AAGTTCTGTAGTACCAAAGC 53 11065 3′UTRCTCCTGGAAGACAGATTCAG 54

[0076] Oligonucleotides ISIS 11061 and 10707 were found to inhibit c-rafRNA levels by greater than 90% in mouse bEND cells at a dose of 400 nM.These two oligonucleotides inhibited raf RNA levels virtually entirelyin rat A-10 cells at a concentration of 200 nM. The IC50 for ISIS 10707was found to be 170 nM in mouse bEND cells and 85 nM in rat A-10 cells.The IC50 for ISIS 11061 was determined to be 85 nM in mouse bEND cellsand 30 nM in rat A-10 cells.

[0077] Effect of ISIS-11061 on Endogenous c-raf mRNA Expression in Mice

[0078] Mice were injected intraperitoneally with ISIS 11061 (50 mg/kg)or control oligonucleotide or saline control once daily for three days.Animals were sacrificed and organs were analyzed for c-raf mRNAexpression by Northern blot analysis. ISIS 11061 was found to decreaselevels of c-raf mRNA in liver by approximately 70%. Controloligonucleotides had no effects on c-raf expression. The effect of ISIS11061 was specific for c-raf; A-raf and G3PDH RNA levels were unaffectedby oligonucleotide treatment.

[0079] Antisense Oligonucleotide to c-raf Increases Survival in MurineHeart Allograft Model

[0080] To determine the therapeutic effects of the c-raf antisenseoligonucleotide ISIS 11061 in preventing allograft rejection, thisoligonucleotide was tested for activity in a murine vascularizedheterotopic heart transplant model. Hearts from C57BI10 mice weretransplanted into the abdominal cavity of C3H mice as primaryvascularized grafts essentially as described by Isobe et al.,Circulation 1991, 84, 1246-1255. Oligonucleotides were administered bycontinuous intravenous administration via a 7-day Alzet pump. The meanallograft survival time for untreated mice was 7.83±0.75 days( 7, 7, 8,8, 8, 9 days). Allografts in mice treated for 7 days with 20 mg/kg or 40mg/kg ISIS 11061 all survived at least 11 days (11, 11, 12 days for 20mg/kg dose and >11, >11, >11 days for the 40 mg/kg dose).

[0081] In a pilot study conducted in rats, hearts from Lewis rats weretransplanted into the abdominal cavity of ACI rats. Rats were dosed withISIS 11061 at 20 mg/kg for 7 days via Alzet pump. The mean allograftsurvival time for untreated rats was 8.86±0.69 days (8, 8, 9, 9, 9, 9,10 days). In rats treated with oligonucleotide, the allograft survivaltime was 15.3±1.15 days (14, 16, 16 days).

[0082] Effects of Antisense Oligonucleotide Targeted to c-raf on SmoothMuscle Cell Proliferation

[0083] Smooth muscle cell proliferation is a cause of blood vesselstenosis, for example in atherosclerosis and restenosis afterangioplasty. Experiments were performed to determine the effect of ISIS11061 on proliferation of A-10 rat smooth muscle cells. Cells in culturewere grown with and without ISIS 11061 (plus lipofectin) and cellproliferation was measured 24 and 48 hours after stimulation with fetalcalf serum. ISIS 11061 (500 nM) was found to inhibit serum-stimulatedcell growth in a dose-dependent manner with a maximal inhibition of 46%and 75% at 24 hours and 48 hours, respectively. An IC50 value of 200 nMwas obtained for this compound. An unrelated control oligonucleotide hadno effect at doses up to 500 nM.

[0084] Effects of Antisense Oligonucleotides Targeted to c-raf onRestenosis in Rats

[0085] A rat carotid artery injury model of angioplasty restenosis hasbeen developed and has been used to evaluate the effects on restenosisof antisense oligonucleotides targeted to the c-myc oncogene. Bennett etal., J. Clin. Invest. 1994, 93, 820-828. This model will be used toevaluate the effects of antisense oligonucleotides targeted to ratc-raf, particularly ISIS 11061, on restenosis. Following carotid arteryinjury with a balloon catheter, oligonucleotides are administered eitherby intravenous injection, continuous intravenous administration viaAlzet pump, or direct administration to the carotid artery in a pluronicgel matrix as described by Bennett et al. After recovery, rats aresacrificed, carotid arteries are examined by microscopy and effects oftreatment on luminal cross-sections are determined.

[0086] Effects of ISIS 5132 (Antisense Oligodeoxynucleotide Targeted toHuman c-raf on Tumor Growth in Human Patients

[0087] Two clinical trials were undertaken to test ISIS 5132 on avariety of human tumors. In one study the compound was administered byintravenous infusion over 2 hours. In the other trial the drug wasadministered by intravenous infusion over 21 days using a continuouspump. Two patients, both of whom had demonstrated tumor progression withprevious cytotoxic chemotherapy, exhibited long-term stable disease inresponse to ISIS 5132 treatment in the 2-hour infusion study (29patients evaluated). In these responding patients levels of c-rafexpression in peripheral blood cells paralleled clinical response. Sixpatients showed stabilization of disease of two months or greater inresponse to ISIS 5132 treatment in the 21-day continuous infusion study(34 patients evaluated). These results are discussed hereinbelow inExamples 13-15.

[0088] The invention is further illustrated by the following exampleswhich are illustrations only and are not intended to limit the presentinvention to specific embodiments.

EXAMPLES Example 1

[0089] Synthesis and Characterization of Oligonucleotides

[0090] Unmodified DNA oligonucleotides were synthesized on an automatedDNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution ofH-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.2′-O-methyl phosphorothioate oligonucleotides were synthesized using2′-O-methyl β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, NeedhamMass.) and the standard cycle for unmodified oligonucleotides, exceptthe wait step after pulse delivery of tetrazole and base was increasedto 360 seconds. The 3′-base used to start the synthesis was a2′-deoxyribonucleotide. 2′-O-propyl oligonucleotides were prepared by aslight modification of this procedure.

[0091] 2′-fluoro phosphorothioate oligonucleotides were synthesizedusing 5′-dimethoxytrityl-3′-phosphoramidites and prepared as disclosedin U.S. patent application Ser. No. 463,358, filed Jan. 11, 1990, and566,977, filed Aug. 13, 1990, which are assigned to the same assignee asthe instant application and which are incorporated by reference herein.The 2′-fluoro oligonucleotides were prepared using phosphoramiditechemistry and a slight modification of the standard DNA synthesisprotocol: deprotection was effected using methanolic ammonia at roomtemperature.

[0092] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

Example 2

[0093] Northern Blot Analysis of Inhibition of c-raf mRNA Expression

[0094] The human urinary bladder cancer cell line T24 was obtained fromthe American Type Culture Collection (Rockville Md.). Cells were grownin McCoy's 5A medium with L-glutamine (Gibco BRL, Gaithersburg Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEMreduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide withlipofectin was then added to the desired concentration. After 4 hours oftreatment, the medium was replaced with McCoy's medium. Cells wereharvested 24 to 72 hours after oligonucleotide treatment and RNA wasisolated using a standard CsCl purification method. Kingston, R. E., inCurrent Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.),John Wiley and Sons, NY. Total RNA was isolated by centrifugation ofcell lysates over a CsCl cushion. RNA samples were electrophoresedthrough 1.2% agarose-formaldehyde gels and transferred to hybridizationmembranes by capillary diffusion over a 12-14 hour period. The RNA wascross-linked to the membrane by exposure to UV light in a Stratalinker(Stratagene, La Jolla, Calif.) and hybridized to random-primed³²P-labeled c-raf cDNA probe (obtained from ATCC) or G3PDH probe as acontrol. RNA was quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

Example 3

[0095] Specific Inhibition of c-raf Kinase Protein Expression in T24Cells

[0096] T24 cells were treated with oligonucleotide (200 nM) andlipofectin at T=0 and T=24 hours. Protein extracts were prepared at T=48hours, electrophoresed on acrylamide gels and analyzed by Western blotusing polyclonal antibodies against c-raf (UBI, Lake Placid, N.Y.) orA-raf (Transduction Laboratories, Knoxville, Tenn.). Radiolabeledsecondary antibodies were used and raf protein was quantitated using aPhosphorimager (Molecular Dynamics, Sunnyvale Calif.).

Example 4

[0097] Antisense Inhibition of Cell Proliferation

[0098] T24 cells were treated on day 0 for two hours with variousconcentrations of oligonucleotide and lipofectin (50 nM oligonucleotidein the presence of 2 μg/ml lipofectin; 100 nM oligonucleotide and 2μg/ml lipofectin; 250 nM oligonucleotide and 6 μg/ml lipofectin or 500nM oligonucleotide and 10 μg/ml lipofectin). On day 1, cells weretreated for a second time at desired oligonucleotide concentration fortwo hours. On day 2, cells were counted.

Example 5

[0099] Effect of ISIS 5132 on T24 Human Bladder Carcinoma TumorXenografts in Nude Mice

[0100] 5×10⁶ T24 cells were implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) were administeredthree times weekly beginning on day 4 after tumor cell inoculation. Asaline-only control was also given. Oligonucleotides were given byintraperitoneal injection. Oligonucleotide dosage was 25 mg/kg. Tumorsize was measured and tumor volume was calculated on the eleventh,fifteenth and eighteenth treatment days.

Example 6

[0101] Diagnostic Assay for raf-associated Tumors Using Xenografts inNude Mice

[0102] Tumors arising from raf expression are diagnosed anddistinguished from other tumors using this assay. A biopsy sample of thetumor is treated, e.g., with collagenase or trypsin or other standardmethods, to dissociate the tumor mass. 5×10⁶ tumor cells are implantedsubcutaneously in the inner thighs of two or more nude mice. Antisenseoligonucleotide (e.g., ISIS 5132) suspended in saline is administered toone or more mice by intraperitoneal injection three times weeklybeginning on day 4 after tumor cell inoculation. Saline only is given toa control mouse. Oligonucleotide dosage is 25 mg/kg. Tumor size ismeasured and tumor volume is calculated on the eleventh treatment day.Tumor volume of the oligonucleotide-treated mice is compared to that ofthe control mouse. The volume of raf-associated tumors in the treatedmice are measurably smaller than tumors in the control mouse. Tumorsarising from causes other than raf expression are not expected torespond to the oligonucleotides targeted to raf and, therefore, thetumor volumes of oligonucleotide-treated and control mice areequivalent.

Example 7

[0103] Detection of raf Expression

[0104] Oligonucleotides are radiolabeled after synthesis by ³²P labelingat the 5′ end with polynucleotide kinase. Sambrook et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989,Volume 2, pg. 11.31-11.32. Radiolabeled oligonucleotides are contactedwith tissue or cell samples suspected of raf expression, such as tumorbiopsy samples or skin samples where psoriasis is suspected, underconditions in which specific hybridization can occur, and the sample iswashed to remove unbound oligonucleotide. Radioactivity remaining in thesample indicates bound oligonucleotide and is quantitated using ascintillation counter or other routine means.

[0105] Radiolabeled oligonucleotides of the invention are also used inautoradiography. Tissue sections are treated with radiolabeledoligonucleotide and washed as described above, then exposed tophotographic emulsion according to standard autoradiography procedures.The emulsion, when developed, yields an image of silver grains over theregions expressing raf. The extent of raf expression is determined byquantitation of the silver grains.

[0106] Analogous assays for fluorescent detection of raf expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorimeter or fluorescence microscope is used to detect thefluorescence which indicates raf expression.

Example 8

[0107] Effect of Oligonucleotide on Endogenous c-raf Expression

[0108] Mice were treated by intraperitoneal injection at anoligonucleotide dose of 50 mg/kg on days 1, 2 and 3. On day 4 animalswere sacrificed and organs removed for c-raf mRNA assay by Northern blotanalysis. Four groups of animals were employed: 1) no oligonucleotidetreatment (saline); 2) negative control oligonucleotide ISIS 1082(targeted to herpes simplex virus; 3) negative control oligonucleotide4189 (targeted to mouse protein kinase C-α; 4) ISIS 11061 targeted torodent c-raf.

Example 9

[0109] Cardiac Allograft Rejection Model

[0110] Hearts were transplanted into the abdominal cavity of rats ormice (of a different strain from the donor) as primary vascularizedgrafts essentially as described by Isobe et al., Circulation 1991, 84,1246-1255. Oligonucleotides were administered by continuous intravenousadministration via a 7-day Alzet pump. Cardiac allograft survival wasmonitored by listening for the presence of a second heartbeat in theabdominal cavity.

Example 10

[0111] Proliferation Assay Using Rat A-10 Smooth Muscle Cells

[0112] A10 cells were plated into 96-well plates in Dulbecco's modifiedEagle medium (DMEM)+10% fetal calf serum and allowed to attach for 24hours. Cells were made quiescent by the addition of DMEM+0.2% dialyzedfetal calf serum for an additional 24 hours. During the last 4 hours ofquiescence, cells were treated with ISIS 11061+lipofectin (Gibco-BRL,Bethesda Md.) in serum-free medium. Medium was then removed, replacedwith fresh medium and the cells were stimulated with 10% fetal calfserum. The plates were the placed into the incubator and cell growth wasevaluated by MTS conversion to formozan (Promega cell proliferation kit)at 24 and 48 hours after serum stimulation. A control oligonucleotide,ISIS 1082 (an unrelated oligonucleotide targeted to herpes simplexvirus), was also tested.

Example 11

[0113] Rat Carotid Artery Restenosis Model

[0114] This model has been described by Bennett et al., J. Clin. Invest.1994, 93, 820-828. Intimal hyperplasia is induced by balloon catheterdilatation of the carotid artery of the rat. Rats are anesthetized andcommon carotid artery injury is induced by passage of a balloonembolectomy catheter distended with 20 ml of saline. Oligonucleotidesare applied to the adventitial surface of the arterial wall in apluronic gel solution. Oligonucleotides are dissolved in a 0.25%pluronic gel solution at 4° C. (F127, BASF Corp.) at the desired dose.100 μl of the gel solution is applied to the distal third of the commoncarotid artery immediately after injury. Control rats are treatedsimilarly with gel containing control oligonucleotide or nooligonucleotide. The neck wounds are closed and the animals allowed torecover. 14 days later, rats are sacrificed, exsanguinated and thecarotid arteries fixed in situ by perfusion with paraformaldehyde andglutaraldehyde, excised and processed for microscopy. Cross-sections ofthe arteries are calculated.

[0115] In an alternative to the pluronic gel administration procedure,rats are treated by intravenous injection or continuous intravenousinfusion (via Alzet pump) of oligonucleotide.

Example 12

[0116] Additional Oligonucleotides Targeted to Human c-raf Kinase

[0117] The oligonucleotides shown in Table 9 were designed using theGenbank c-raf sequence HSRAFR (Genbank accession no. x03484; SEQ ID NO:64), synthesized and tested for inhibition of c-raf mRNA expression asdescribed in Examples 1 and 2. All are oligodeoxynucleotides withphosphorothioate backbones and all are targeted to the 3′ UTR of humanc-raf. TABLE 9 Human c-raf Kinase Antisense Oligonucleotides Isis #Sequence (5′ → 3′) SEQ ID NO 11459 TTGAGCATGGGGAATGTGGG 55 11457AACATCAACATCCACTTGCG 56 11455 TGTAGCCAACAGCTGGGGCT 57 11453CTGAGAGGGCTGAGATGCGG 58 11451 GCTCCTGGAAGACAAAATTC 59 11449TGTGACTAGAGAAACAAGGC 60 11447 CAAGAAAACCTGTATTCCTG 61 11445TTGTCAGGTGCAATAAAAAC 62 11443 TTAAAATAACATAATTGAGG 63

[0118] Of these, ISIS 11459 and 11449 gave 38% and 31% inhibition ofc-raf mRNA levels in this assay and are, therefore, preferred. ISIS11451, 11445 and 11443 gave 18%, 11% and 7% inhibition of c-rafexpression, respectively.

Example 13

[0119] Effect of Antisense Oligonucleotide Targeted to c-raf on Patientswith Cancer—2 Hour Infusion

[0120] Twenty-nine fully evaluable patients with a range of cancer typesreceived ISIS 5132 as a two-hour infusion three times weekly for threeweeks. Following a one-week treatment-free interval, treatment wasresumed, and maintained as long as the patient remained free of tumorprogression or significant toxicity. Doses were escalated from 0.5 to6.0 mg/kg in cohorts of three patients. The drug was well-tolerated andno patient required dose reduction.

[0121] Patients with refractory malignancies received ISIS 5132 at2-hour intravenous infusion three times weekly for 3 consecutive weeksat one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg.Eligibility required adequate bone marrow function (neutrophils≧1,5000/mm³, hemoglobin ≧9.0 g/dL, and platelets ≧1000,000/mm³), serumcreatine <2.0 mg/dL, total bilirubin <2.0 mg/dL, aspartateaminotransferase <2 times upper normal limit (<5 times upper normallimit in the presence of liver metastases), and no prolongation of theprothrombin time (PT) or activated partial thromboplastin time (aPTT).Blood counts and biochemical profiles were performed twice weekly duringthe first week and once a week thereafter. ISIS 5132 was supplied as asterile solution in vials containing 1.1 mL or 10.5 mL ofphosphate-buffered saline at a concentration of 10 mg/mL. Prior toadministration, ISIS 5132 was diluted in normal saline to a total volumeof 50 mL and the infused intravenously over two hours. Following aone-week treatment-free interval, dosing was resumed and maintained aslong as the patient remained free of tumor progression or significanttoxicity.

Example 14

[0122] Reduction of c-raf Expression in Peripheral Blood MononuclearCells of Cancer Patients After Treatment with Antisense Oligonucleotide

[0123] Peripheral-blood mononuclear cells (PBMCS) for c-raf mRNAanalysis were collected at baseline and on days 3, 5, 8, and 15 of cycle1 and on day 1 of each cycle thereafter. PBMCs were isolated byFicoll-Hypaque density centrifugation and stored at −70° C. Total RNAwas isolated using Trizol reagent (Gibco BRL, Rockville, Md.) accordingto the manufacturer's directions. Because of the low abundance of thec-raf message in PBMCs, mRNA quantitation was performed using areverse-transcriptase polymerase chain reaction (RT-PCR) assay. 100 ngtotal RNA was used for each cDNA reaction. C-raf expression wasnormalized to that of the endogenous standard β-actin by calculating theration of the radiolabeled PCR products. PCR reactions (25 ul totalvolume, containing 0.1-10 μl cDNA, 12.5 pmol of each of the c-raf orβ-actin primers, and 1 μCi α-³²P dCTP) were heated to 95° C. for 5minutes then amplified for 28-36 cycles at 95° C. for 1 minute, 55° C.for 1 minute and 72° C. for 2 minutes. The products were loaded on 8%urea polyacrylamide gels which were then dried at 80° C. for 1 hourunder vacuum and exposed to film for several hours at −80° C. Reductionsin c-raf expression were identified in 13 of 14 patients within 48 hoursof initial ISIS 5132 dosing. The median reduction was to 42% (mean 53%)of initial values (p=0.002). Compared to baseline values, medianreduction in expression on day 5 was 26% (mean 71%; p=0.017), on day 832% (mean 81%; p=0.03), and on day 15 35% (mean 74%; p=0.017).

[0124] Clinical Responses in Cancer Patients—2 hr Infusion:

[0125] Two patients, both of whom had demonstrated tumor progressionwith previous cytotoxic chemotherapy, exhibited long-term stable diseasein response to ISIS 5132 treatment. One was a 68-year old man withcolorectal cancer metastatic to liver who had progressed two years afteradjuvant therapy with 5-fluorouracil/leucovorin, and had evinced furthertumor growth during therapy with a 17-1A monoclonal antibody andirinotecan. Following treatment with 3 mg/kg of ISIS 5132, minor (20%)shrinkage in a liver metastasis was accompanied by a progressive declinein choreoembryonic antigen (CEA, a marker for colon cancer) from 895ng/mL to 618 ng/mL. During this time, c-raf mRNA values declined tobelow 10% of the initial value. After seven cycles of treatment, boththe plasma CEA values and the PBMC c-raf mRNA began to increase, and onemonth later a CT scan revealed progression of the hepatic metastases.

[0126] A 46-year old woman with renal cell cancer metastatic to lung andlymph nodes failed to respond to interleukin-2, α-interferon and5-fluorouracil in combination, and began treatment with ISIS 5132 at 5mg/kg. She had immediate symptomatic improvement, but the size of thetumor was unchanged on CT scans. After ten cycles of treatment, shebegan to have recurrent pain, and progression was identifiedradiologically. In this patient the nadir PBMC c-raf mRNA was 9%, andvalues remained low until the beginning of the ninth cycle, when areturn above baseline was observed, again followed shortly thereafter byprogressive disease.

Example 15

[0127] Effect of Antisense Oligonucleotide Targeted to c-raf on Patientswith Cancer—21 Day Continuous Infusion

[0128] A continuous intravenous infusion of ISIS 5132 was administeredfor 21 days every 4 weeks to 34 patients with a variety of solid tumorsrefractory to standard therapy. The dose of ISIS 5132 was increased insequential cohorts of patients, as toxicity allowed, until a final doseof 5.0 mg/kg of body weight was reached.

[0129] Eligible patients had histologically-documented solidmalignancies of measurable or evaluable status refractory to standardtherapy or for whom no effective therapy existed. Patients wereprescreened in regard to their medical history as described above withthe addition of the measurement of complement split products prior tothe first infusion of ISIS 5132, 4 and 24 hours after starting theinfusion and, repeated on days 7, 14 and 21. Patients receivedsequential, ascending, multiple doses of ISIS 5132 administered as acontinuous IV infusion for 21 consecutive days at a pump rate of 1.5mL/hour followed by one week of rest (one cycle). The initial dose ofISIS 5231 was 0.5 mg/kg of body weight. Subsequent doses were 1.0, 1.5,2.0, 3.0, 4.0, and 5.0 mg/kg. The total dose was added to 250 mL ofnormal saline and infused as described above.

[0130] Clinical Responses in Cancer Patients—22-day Infusion:

[0131] Six patients showed stabilization of disease of two months orgreater. Of these two patients had prolonged stabilization: one patient(treated at 1.5 mg/kg/day) with renal cell carcinoma remained stable for9 months, and the other (treated at 4.0 mg/kg/day) with pancreaticcancer remained stable for 10 months. The most significant responseoccurred in a 57-year old female with ovarian cancer, treated at 3.0mg/kg/day. Her CA-125 level (a marker for ovarian cancer) at the time ofinitial surgical resection was 3300 u/mL. Following resection and abrief course of taxol and platinum, her CA-125 level was reportedlynormal, but began to markedly increase again within 8 months. She wasthen treated with a succession of systemic therapies, most of whichachieved only a short term, modest decrease in CA-125 levels. At thetime of initiation of ISIS 5132 infusions, her CA-125 level was 1490u/mL. She was treated with 10 cycles of ISIS 5132 and achieved a 97%reduction in tumor marker levels.

Example 16

[0132] Effect of Antisense Oligonucleotide Targeted to c-raf (21 DayInfusion) in Combination with Other Chemotherapeutic Agents in CancerPatients

[0133] Fourteen patients with refractory cancers were given ISIS 5132 atdoses of 1.0-3.0 mg/kg/day as a 21 day IV infusion in combination with5-fluorouracil (425 mg/m²) and Leucovorin (20 mg/m²) as an IV bolusgiven on days 1-5 every 4 weeks. In this ongoing study, 8 patients havebeen treated at the 2.0 mg/kg/day dose level. Toxicities that occurredwere not dose-limiting. Disease stabilization lasting at least 4 cyclesoccurred in 4 patients (2 renal cell, 1 colon, 1 pancreatic). Thus ISIS5132 at a dose of 2 mg/kg/day is active and well tolerated incombination with 5-FU/LV on this schedule.

Example 17

[0134] Effect of Antisense Oligonucleotide Targeted to c-raf in PigBranch Retinal Vein Occlusion Model of Ocular Neovascularization

[0135] Angiogenesis, or neovascularization, is the formation of newcapillaries from existing blood vessels. In adult organisms this processis typically controlled and short-lived, for example in wound repair andregeneration. Gaiso, M. L., 1999, Medscape Oncology 2(1), Medscape Inc.However, aberrant capillary growth can occur and this uncontrolledgrowth plays a causal and/or supportive role in many pathologicconditions such as tumor growth and metastasis. In the context of thisinvention “aberrant angiogenesis” refers to unwanted or uncontrolledangiogenesis. Angiogenesis inhibitors are being evaluated for use asantitumor drugs. Other diseases and conditions associated withangiogenesis include arthritis, cardiovascular diseases, skinconditions, and aberrant wound healing. Aberrant angiogenesis can alsooccur in the eye, causing loss of vision. Examples of ocular conditionsinvolving aberrant angiogenesis include macular degeneration, diabeticretinopathy and retinopathy of prematurity. A pig model of ocularneovascularization, the branch retinal vein occlusion (BVO) model, isused to study ocular neovascularization. An antisense oligonucleotidetargeted to pig c-raf, ISIS 107189 (CCACACCACTCATCTCATCT; SEQ ID NO: 66)was tested in this model.

[0136] Male farm pigs (8-10 kg) were subjected to branch retinal veinocclusions (BVO) by laser treatment in both eyes. The extent of BVO wasdetermined by indirect opthalmoscopy after a 2 week period.Intravitreous injections (10 μM) of ISIS 107189 were started on the dayof BVO induction and were repeated at weeks 2, 6, and 10 after BVO(Right eye—vehicle, Left eye—antisense molecule). Stereo fundusphotography and fluorescein angiography were performed at baseline BVOand at weeks 1,6 and 12 following intravitreous injections. In additioncapillary gel electrophoresis analysis of the eye sections containingsclera, choroid, and the retina were performed to determine antisenseconcentrations, and gross and microscopic evaluations were performed todetermine eye histopathology.

[0137] The antisense oligonucleotide targeted to c-raf significantlyinhibited the neovascularization response compared to vehicle-onlyinjections (p=0.05).

Example 18

[0138] Oligonucleotide Inhibition of B-raf Expression

[0139] The oligonucleotides shown in Table 10 were designed using theGenbank B-raf sequence HUMBRAF (Genbank listings M95712;M95720;x54072),provided herein as SEQ ID NO: 67, synthesized and tested for inhibitionof B-raf mRNA expression in T24 bladder carcinoma cells or A549 lungcarcinoma cells using a Northern blot assay. The human urinary bladdercancer cell line T24 and the human lung tumor cell line A549 wereobtained from the American Type Culture Collection (Rockville Md.). T24cells were grown in McCoy's 5A medium with L-glutamine and A549 cellswere grown in DMEM low glucose medium (Gibco BRL, Gaithersburg Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEMreduced-serum medium containing 2.5 μl DOTMA per 100 nM oligonucleotide.Oligonucleotide with lipofectin was then added to the desiredconcentration. After 4 hours of treatment, the medium was replaced withappropriate medium (McCoy's or DMEM low glucose). Cells were harvested24 to 72 hours after oligonucleotide treatment and RNA was isolatedusing a standard CsCl purification method. Kingston, R. E., in CurrentProtocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.),John Wiley and Sons, NY. Total RNA was isolated by centrifugation ofcell lysates over a CsCl cushion. RNA samples were electrophoresedthrough 1.2% agarose-formaldehyde gels and transferred to hybridizationmembranes by capillary diffusion over a 12-14 hour period. The RNA wascross-linked to the membrane by exposure to UV light in a Stratalinker(Stratagene, La Jolla, Calif.) and hybridized to a ³²P-labeled B-rafcDNA probe or G3PDH probe as a control. The human B-raf cDNA probe wascloned by PCR using complementary oligonucleotide primers after reversetranscription of total RNA. Identity of the B-raf cDNA was confirmed byrestriction digestion and direct DNA sequencing. RNA was quantitatedusing a Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.). TABLE 10Human B-raf Kinase Antisense Oligonucleotides (All are phosphorothioateoligodeoxynucleotides) Isis # Sequence (5′ → 3′) Site SEO ID NO: 13720ATTTTGAAGGAGACGGACTG coding 68 13721 TGGATTTTGAAGGAGACGGA coding 6913722 CGTTAGTTAGTGAGCCAGGT coding 70 13723 ATTTCTGTAAGGCTTTCACG coding71 13724 CCCGTCTACCAAGTGTTTTC coding 72 13725 AATCTCCCAATCATCACTCGcoding 73 13726 TGCTGAGGTGTAGGTGCTGT coding 74 13727TGTAACTGCTGAGGTGTAGG coding 75 13728 TGTCGTGTTTTCCTGAGTAC coding 7613729 AGTTGTGGCTTTGTGGAATA coding 77 13730 ATGGAGATGGTGATACAAGC coding78 13731 GGATGATTGACTTGGCGTGT coding 79 13732 AGGTCTCTGTGGATGATTGAcoding 80 13733 ATTCTGATGACTTCTGGTGC coding 81 13734GCTGTATGGATTTTTATCTT coding 82 13735 TACACAACAATCCCAAATGC coding 8313736 ATCCTCGTCCCACCATAAAA coding 84 13737 CTCTCATCTCTTTTCTTTTT coding85 13738 GTCTCTCATCTCTTTTCTTT coding 86 13739 CCGATTCAAGGAGCGTTCTGcoding 87 13740 TGGATGGGTGTTTTTGGAGA coding 88 13741CTGCCTGGATGGGTGTTTTT coding 89 14144 GGACAGGAAACGCACCATAT coding 9014143 CTCATTTGTTTCAGTGGACA stop codon 91 14142 TCTCTCACTCATTTGTTTCA stopcodon 92 14141 ACTCTCTCACTCATTTGTTT stop codon 93 14140GAACTCTCTCACTCATTTGT coding 94 14139 TCCTGAACTCTCTCACTCAT coding 9514138 TTGCTACTCTCCTGAACTCT coding 96 14137 TTTGTTGCTACTCTCCTGAG coding97 14136 CTTTTGTTGCTACTCTCCTG coding 98 13742 GCTACTCTCCTGAACTCTCTcoding 99 14135 TTCCTTTTGTTGCTACTCTC coding 100 14134ATTTATTTTCCTTTTGTTGC coding 101 14133 ATATGTTCATTTATTTTCCT coding 10213743 TTTATTTTCCTTTTGTTGCT coding 103 13744 TGTTCATTTATTTTCCTTTT coding104 14132 ATTTAACATATAAGCAAACA coding 105 14529 CTGCCTGGTACCCTGTTTTT 5mismatch 106 14530 CTGCCTGGAAGGGTGTTTTT 1 mismatch 107 14531CTGCCTGGTACGGTGTTTTT 3 mismatch 108

[0140] There are multiple B-raf transcripts. The two most prevalenttranscripts were quantitated after oligonucleotide treatment. Thesetranscripts run at approximately 8.5 kb (upper transcript) and 4.7 kb(lower transcript) under the gel conditions used. Both transcripts aretranslated into B-raf protein in cells. In the initial screen, A549cells were treated with oligonucleotides at a concentration of 200 nMoligonucleotide for four hours in the presence of lipofectin. Resultswere normalized and expressed as a percent of control. In this initialscreen, oligonucleotides giving a reduction of either B-raf mRNAtranscript of approximately 30% or greater were considered active.According to this criterion, oligonucleotides 13722, 13724, 13726,13727, 13728, 13730, 13732, 13733, 13736, 13739, 13740, 13741, 13742,13743, 14135, 14136, 14138 and 14144 were found to be active. Thesesequences are therefore preferred. Of these, oligonucleotides 13727,13730, 13740, 13741, 13743 and 14144 showed 40-50% inhibition of one orboth B-raf transcripts in at least one assay. These sequences aretherefore more preferred. In one of the two assays, ISIS 14144 (SEQ IDNO: 23) reduced levels of both transcripts by 50-60% and ISIS 13741 (SEQID NO: 22) reduced both transcripts by 65-70%. These two sequences aretherefore highly preferred.

[0141] Dose response experiments were done in both T24 cells and A549cells for the two most active oligonucleotides, ISIS 13741 and ISIS14144 (SEQ ID NO: 89 and 90), along with mismatch control sequenceshaving 1, 3 or 5 mismatches of the ISIS 13741 sequence. ISIS 13741 and14144 had almost identical activity in this assay when the upper B-raftranscript was measured, with IC50s between 250 and 300 nM. The mismatchcontrols had no activity (ISIS 14531) or slight activity, with a maximuminhibition of less than 20% at the 400 nM dose (ISIS 14530, ISIS 14529).Against the lower B-raf transcript, ISIS 13741 and ISIS 14144 had IC50sof approximately 350 and 275 nM, respectively in this assay, with themismatch controls never achieving 50% inhibition at concentrations up to400 nM. Therefore, ISIS 13741 and 14144 are preferred.

[0142] Reduction of B-raf mRNA levels was measured in T24 cells by theseoligonucleotides (all are phosphorothioate oligodeoxynucleotides) aftera 4-hour treatment in the presence of lipofectin. Results are normalizedto G3PDH and expressed as a percent of control. Against the uppertranscript, ISIS 13741 and 14144 were again most active, with IC50s ofapproximately 100 nM and 275 nM, respectively, in this assay. Themismatch controls 14529 and 14531 had no activity, and the mismatchcontrol 14530 achieved a maximum reduction of raf mRNA of approximately20% at a 400 nM dose. Against the lower transcript, ISIS 13741 had anIC50 of approximately 100-125 nM and ISIS 14144 had an IC50 ofapproximately 250 nM in this assay, with the mismatch controlscompletely inactive. Therefore ISIS 13741 and 14144 are preferred.

[0143] 2′-Methoxyethoxy (2′-MOE) Oligonucleotides Targeted to B-raf:

[0144] The oligonucleotides shown in Table 11 were synthesized.Nucleotides shown in bold are 2′-MOE. 2′-MOE cytosines are all5-methylcytosines. For backbone linkage, “s” indicates phosphorothioate(P═S) and “o” indicates phosphodiester (P═O). TABLE 11 2′-MOEoligonucleotides targeted to human B-raf (bold = 2′-MOE) SEQ ID ISIS#Sequence/modification NO: 13741 CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT89 15339 CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 89 15340CoToGoCoCoToGoGoAoToGsGsGsTsGsTsTsTsTsT 89 15341CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 89 15342CoToGoCoCsTsGsGsAsTsGsGsGsTsGoToToToToT 89 15343CsTsGsCsCsTsGsGsAsToGoGoGoToGoToToToToT 89 15344CsTsGsCsCsTsGsGsAsTsGsGsGsTsGsTsTsTsTsT 89

[0145] These oligonucleotides were tested for their ability to reduceB-raf mRNA levels in T24 cells. Against the lower transcript, ISIS 13741(P═S deoxy) and ISIS 15344 (P═S deoxy/MOE) had IC50s of approximately250 nM. The other two compounds tested, ISIS 15341 and 15342, did notachieve 50% inhibition at doses up to 400 nM. Against the uppertranscript, ISIS 13741 and 15344 demonstrated IC50s of approximately 150nM, ISIS 15341 demonstrated an IC50 of approximately 200 nM and ISIS15342 did not achieve 50% reduction at doses up to 400 nM. Based onthese results, ISIS 15341, 13741 and 15344 are preferred.

1 130 1 20 DNA artificial sequence antisense sequence 1 tgaaggtgagctggagccat 20 2 20 DNA artificial sequence antisense sequence 2gctccattga tgcagcttaa 20 3 20 DNA artificial sequence antisense sequence3 ccctgtatgt gctccattga 20 4 20 DNA artificial sequence antisensesequence 4 ggtgcaaagt caactagaag 20 5 20 DNA artificial sequenceantisense sequence 5 attcttaaac ctgagggagc 20 6 20 DNA artificialsequence antisense sequence 6 gatgcagctt aaacaattct 20 7 20 DNAartificial sequence antisense sequence 7 cagcactgca aatggcttcc 20 8 20DNA artificial sequence antisense sequence 8 tcccgcctgt gacatgcatt 20 920 DNA artificial sequence antisense sequence 9 gccgagtgcc ttgcctggaa 2010 20 DNA artificial sequence antisense sequence 10 agagatgcagctggagccat 20 11 20 DNA artificial sequence antisense sequence 11aggtgaaggc ctggagccat 20 12 20 DNA artificial sequence antisensesequence 12 gtctggcgct gcaccactct 20 13 20 DNA artificial sequenceantisense sequence 13 ctgatttcca aaatcccatg 20 14 20 DNA artificialsequence antisense sequence 14 ctgggctgtt tggtgcctta 20 15 20 DNAartificial sequence antisense sequence 15 tcagggctgg actgcctgct 20 16 20DNA artificial sequence antisense sequence 16 ggtgagggag cgggaggcgg 2017 20 DNA artificial sequence antisense sequence 17 cgctcctcctccccgcggcg 20 18 20 DNA artificial sequence antisense sequence 18ttcggcggca gcttctcgcc 20 19 20 DNA artificial sequence antisensesequence 19 gccgccccaa cgtcctgtcg 20 20 20 DNA artificial sequenceantisense sequence 20 tcctcctccc cgcggcgggt 20 21 20 DNA artificialsequence antisense sequence 21 ctcgcccgct cctcctcccc 20 22 20 DNAartificial sequence antisense sequence 22 ctggcttctc ctcctcccct 20 23 20DNA artificial sequence antisense sequence 23 cgggaggcgg tcacattcgg 2024 20 DNA artificial sequence antisense sequence 24 tctggcgctgcaccactctc 20 25 2977 DNA homo sapiens 25 ccgaatgtga ccgcctcccgctccctcacc cgccgcgggg aggaggagcg 50 ggcgagaagc tgccgccgaa cgacaggacgttggggcggc ctggctccct 100 caggtttaag aattgtttaa gctgcatcaa tggagcacatacagggagct 150 tggaagacga tcagcaatgg ttttggattc aaagatgccg tgtttgatgg200 ctccagctgc atctctccta caatagttca gcagtttggc tatcagcgcc 250gggcatcaga tgatggcaaa ctcacagatc cttctaagac aagcaacact 300 atccgtgttttcttgccgaa caagcaaaga acagtggtca atgtgcgaaa 350 tggaatgagc ttgcatgactgccttatgaa agcactcaag gtgaggggcc 400 tgcaaccaga gtgctgtgca gtgttcagacttctccacga acacaaaggt 450 aaaaaagcac gcttagattg gaatactgat gctgcgtctttgattggaga 500 agaacttcaa gtagatttcc tggatcatgt tcccctcaca acacacaact550 ttgctcggaa gacgttcctg aagcttgcct tctgtgacat ctgtcagaaa 600ttcctgctca atggatttcg atgtcagact tgtggctaca aatttcatga 650 gcactgtagcaccaaagtac ctactatgtg tgtggactgg agtaacatca 700 gacaactctt attgtttccaaattccacta ttggtgatag tggagtccca 750 gcactacctt ctttgactat gcgtcgtatgcgagagtctg tttccaggat 800 gcctgttagt tctcagcaca gatattctac acctcacgccttcaccttta 850 acacctccag tccctcatct gaaggttccc tctcccagag gcagaggtcg900 acatccacac ctaatgtcca catggtcagc accacgctgc ctgtggacag 950caggatgatt gaggatgcaa ttcgaagtca cagcgaatca gcctcacctt 1000 cagccctgtccagtagcccc aacaatctga gcccaacagg ctggtcacag 1050 ccgaaaaccc ccgtgccagcacaaagagag cgggcaccag tatctgggac 1100 ccaggagaaa aacaaaatta ggcctcgtggacagagagat tcaagctatt 1150 attgggaaat agaagccagt gaagtgatgc tgtccactcggattgggtca 1200 ggctcttttg gaactgttta taagggtaaa tggcacggag atgttgcagt1250 aaagatccta aaggttgtcg acccaacccc agagcaattc caggccttca 1300ggaatgaggt ggctgttctg cgcaaaacac ggcatgtgaa cattctgctt 1350 ttcatggggtacatgacaaa ggacaacctg gcaattgtga cccagtggtg 1400 cgagggcagc agcctctacaaacacctgca tgtccaggag accaagtttc 1450 agatgttcca gctaattgac attgcccggcagacggctca gggaatggac 1500 tatttgcatg caaagaacat catccataga gacatgaaatccaacaatat 1550 atttctccat gaaggcttaa cagtgaaaat tggagatttt ggtttggcaa1600 cagtaaagtc acgctggagt ggttctcagc aggttgaaca acctactggc 1650tctgtcctct ggatggcccc agaggtgatc cgaatgcagg ataacaaccc 1700 attcagtttccagtcggatg tctactccta tggcatcgta ttgtatgaac 1750 tgatgacggg ggagcttccttattctcaca tcaacaaccg agatcagatc 1800 atcttcatgg tgggccgagg atatgcctccccagatctta gtaagctata 1850 taagaactgc cccaaagcaa tgaagaggct ggtagctgactgtgtgaaga 1900 aagtaaagga agagaggcct ctttttcccc agatcctgtc ttccattgag1950 ctgctccaac actctctacc gaagatcaac cggagcgctt ccgagccatc 2000cttgcatcgg gcagcccaca ctgaggatat caatgcttgc acgctgacca 2050 cgtccccgaggctgcctgtc ttctagttga ctttgcacct gtcttcaggc 2100 tgccagggga ggaggagaagccagcaggca ccacttttct gctccctttc 2150 tccagaggca gaacacatgt tttcagagaagctctgctaa ggaccttcta 2200 gactgctcac agggccttaa cttcatgttg ccttcttttctatccctttg 2250 ggccctggga gaaggaagcc atttgcagtg ctggtgtgtc ctgctccctc2300 cccacattcc ccatgctcaa ggcccagcct tctgtagatg cgcaagtgga 2350tgttgatggt agtacaaaaa gcaggggccc agccccagct gttggctaca 2400 tgagtatttagaggaagtaa ggtagcaggc agtccagccc tgatgtggag 2450 acacatggga ttttggaaatcagcttctgg aggaatgcat gtcacaggcg 2500 ggactttctt cagagagtgg tgcagcgccagacattttgc acataaggca 2550 ccaaacagcc caggactgcc gagactctgg ccgcccgaaggagcctgctt 2600 tggtactatg gaacttttct taggggacac gtcctccttt cacagcttct2650 aaggtgtcca gtgcattggg atggttttcc aggcaaggca ctcggccaat 2700ccgcatctca gccctctcag gagcagtctt ccatcatgct gaattttgtc 2750 ctccaggagctgcccctatg gggcgggccg cagggccagc ctgtttctct 2800 aacaaacaaa caaacaaacagccttgtttc tctagtcaca tcatgtgtat 2850 acaaggaagc caggaataca ggttttcttgatgatttggg ttttaatttt 2900 gtttttattg cacctgacaa aatacagtta tctgatggtccctcaattat 2950 gttattttaa taaaataaat taaattt 2977 26 20 DNA artificialsequence antisense sequence 26 ttctcgcccg ctcctcctcc 20 27 20 DNAartificial sequence antisense sequence 27 ttctcctcct cccctggcag 20 28 20DNA artificial sequence antisense sequence 28 cctgctggct tctcctcctc 2029 2458 DNA homo sapiens 29 tgacccaata agggtggaag gctgagtccc gcagagccaataacgagagt 50 ccgagaggcg acggaggcgg actctgtgag gaaacaagaa gagaggccca 100agatggagac ggcggcggct gtagcggcgt gacaggagcc ccatggcacc 150 tgcccagccccacctcagcc catcttgaca aaatctaagg ctccatggag 200 ccaccacggg gcccccctgccaatggggcc gagccatccc gggcagtggg 250 caccgtcaaa gtatacctgc ccaacaagcaacgcacggtg gtgactgtcc 300 gggatggcat gagtgtctac gactctctag acaaggccctgaaggtgcgg 350 ggtctaaatc aggactgctg tgtggtctac cgactcatca agggacgaaa400 gacggtcact gcctgggaca cagccattgc tcccctggat ggcgaggagc 450tcattgtcga ggtccttgaa gatgtcccgc tgaccatgca caattttgta 500 cggaagaccttcttcagcct ggcgttctgt gacttctgcc ttaagtttct 550 gttccatggc ttccgttgccaaacctgtgg ctacaagttc caccagcatt 600 gttcctccaa ggtccccaca gtctgtgttgacatgagtac caaccgccaa 650 cagttctacc acagtgtcca ggatttgtcc ggaggctccagacagcatga 700 ggctccctcg aaccgccccc tgaatgagtt gctaaccccc cagggtccca750 gcccccgcac ccagcactgt gacccggagc acttcccctt ccctgcccca 800gccaatgccc ccctacagcg catccgctcc acgtccactc ccaacgtcca 850 tatggtcagcaccacggccc ccatggactc caacctcatc cagctcactg 900 gccagagttt cagcactgatgctgccggta gtagaggagg tagtgatgga 950 accccccggg ggagccccag cccagccagcgtgtcctcgg ggaggaagtc 1000 cccacattcc aagtcaccag cagagcagcg cgagcggaagtccttggccg 1050 atgacaagaa gaaagtgaag aacctggggt accgggantc aggctattac1100 tgggaggtac cacccagtga ggtgcagctg ctgaagagga tcgggacggg 1150ctcgtttggc accgtgtttc gagggcggtg gcatggcgat gtggccgtga 1200 aggtgctcaaggtgtcccag cccacagctg agcaggccca ggctttcaag 1250 aatgagatgc aggtgctcaggaagacgcga catgtcaaca tcttgctgtt 1300 tatgggcttc atgacccggc cgggatttgccatcatcaca cagtggtgtg 1350 agggctccag cctctaccat cacctgcatg tggccgacacacgcttcgac 1400 atggtccagc tcatcgacgt ggcccggcag actgcccagg gcatggacta1450 cctccatgcc aagaacatca tccaccgaga tctcaagtct aacaacatct 1500tcctacatga ggggctcacg gtgaagatcg gtgactttgg cttggccaca 1550 gtgaagactcgatggagcgg ggcccagccc ttggagcagc cctcaggatc 1600 tgtgctgtgg atggcagctgaggtgatccg tatgcaggac ccgaacccct 1650 acagcttcca gtcagacgtc tatgcctacggggttgtgct ctacgagctt 1700 atgactggct cactgcctta cagccacatt ggctgccgtgaccagattat 1750 ctttatggtg ggccgtggct atctgtcccc ggacctcagc aaaatctcca1800 gcaactgccc caaggccatg cggcgcctgc tgtctgactg cctcaagttc 1850cagcgggagg agcggcccct cttcccccag atcctggcca caattgagct 1900 gctgcaacggtcactcccca agattgagcg gagtgcctcg gaaccctcct 1950 tgcaccgcac ccaggccgatgagttgcctg cctgcctact cagcgcagcc 2000 cgccttgtgc cttaggcccc gcccaagccaccagggagcc aatctcagcc 2050 ctccacgcca aggagccttg cccaccagcc aatcaatgttcgtctctgcc 2100 ctgatgctgc ctcaggatcc cccattcccc accctgggag atgagggggt2150 ccccatgtgc ttttccagtt cttctggaat tgggggaccc ccgccaaaga 2200ctgagccccc tgtctcctcc atcatttggt ttcctcttgg ctttggggat 2250 acttctaaattttgggagct cctccatctc caatggctgg gatttgtggc 2300 agggattcca ctcagaacctctctggaatt tgtgcctgat gtgccttcca 2350 ctggattttg gggttcccag caccccatgtggattttggg gggtcccttt 2400 tgtgtctccc ccgccattca aggactcctc tctttcttcaccaagaagca 2450 cagaattc 2458 30 20 DNA artificial sequence antisensesequence 30 gtcaagatgg gctgaggtgg 20 31 20 DNA artificial sequenceantisense sequence 31 ccatcccgga cagtcaccac 20 32 20 DNA artificialsequence antisense sequence 32 atgagctcct cgccatccag 20 33 20 DNAartificial sequence antisense sequence 33 aatgctggtg gaacttgtag 20 34 20DNA artificial sequence antisense sequence 34 ccggtacccc aggttcttca 2035 20 DNA artificial sequence antisense sequence 35 ctgggcagtctgccgggcca 20 36 20 DNA artificial sequence antisense sequence 36cacctcagct gccatccaca 20 37 20 DNA artificial sequence antisensesequence 37 gagattttgc tgaggtccgg 20 38 20 DNA artificial sequenceantisense sequence 38 gcactccgct caatcttggg 20 39 20 DNA artificialsequence antisense sequence 39 ctaaggcaca aggcgggctg 20 40 20 DNAartificial sequence antisense sequence 40 acgaacattg attggctggt 20 41 20DNA artificial sequence antisense sequence 41 gtatccccaa agccaagagg 2042 20 DNA artificial sequence antisense sequence 42 catcagggcagagacgaaca 20 43 20 DNA artificial sequence antisense sequence 43ggaacatctg gaatttggtc 20 44 20 DNA artificial sequence antisensesequence 44 gattcactgt gacttcgaat 20 45 20 DNA artificial sequenceantisense sequence 45 gcttccattt ccagggcagg 20 46 20 DNA artificialsequence antisense sequence 46 aagaaggcaa tatgaagtta 20 47 20 DNAartificial sequence antisense sequence 47 gtggtgcctg ctgactcttc 20 48 20DNA artificial sequence antisense sequence 48 ctggtggcct aagaacagct 2049 20 DNA artificial sequence antisense sequence 49 gtatgtgctccattgatgca 20 50 20 DNA artificial sequence antisense sequence 50tccctgtatg tgctccattg 20 51 20 DNA artificial sequence antisensesequence 51 atacttatac ctgagggagc 20 52 20 DNA artificial sequenceantisense sequence 52 atgcattctg cccccaagga 20 53 20 DNA artificialsequence antisense sequence 53 gacttgtata cctctggagc 20 54 20 DNAartificial sequence antisense sequence 54 actggcactg caccactgtc 20 55 20DNA artificial sequence antisense sequence 55 aagttctgta gtaccaaagc 2056 20 DNA artificial sequence antisense sequence 56 ctcctggaagacagattcag 20 57 20 DNA artificial sequence antisense sequence 57ttgagcatgg ggaatgtggg 20 58 20 DNA artificial sequence antisensesequence 58 aacatcaaca tccacttgcg 20 59 20 DNA artificial sequenceantisense sequence 59 tgtagccaac agctggggct 20 60 20 DNA artificialsequence antisense sequence 60 ctgagagggc tgagatgcgg 20 61 20 DNAartificial sequence antisense sequence 61 gctcctggaa gacaaaattc 20 62 20DNA artificial sequence antisense sequence 62 tgtgactaga gaaacaaggc 2063 20 DNA artificial sequence antisense sequence 63 caagaaaacctgtattcctg 20 64 20 DNA artificial sequence antisense sequence 64ttgtcaggtg caataaaaac 20 65 20 DNA artificial sequence antisensesequence 65 ttaaaataac ataattgagg 20 66 2018 DNA sus scrofa 66tcgaattcga agtcacagtg aatcagcctc accttcagcc ttgtccagca gccccaacaa 60tctgagccca acctgggtca caaccgaaaa cccctgtgcc agcacagaga gagcgggcgc 120caggatccgg gacccaggag aaaaacaaaa ttaggcctcg tggacagaga gattcaagct 180attactggga aatagaagcc agtgaagtga tgctttccac tcggattggg tcaggctcct 240ttggaactgt ttatagggca agtggcatgg agatgttgca gtaaagatcc taaaggttgt 300tgaccccaca ccagagcagt tgcaggcctt taggaatgaa gtggctgtcc ttcgcaaaac 360tcggcatgtg aacatcctgc tgttcatggg gtacatgacc aaggacaacc tggcgattgt 420gacccagtgg tgtgagggca gcagcctcta caaacacctg catgtccagg agaccaagtt 480ccagatgttc cagttgattg acattgcccg gcagacggct cagggaatgg actacttgca 540tgcaaagaac atcatccaca gagacatgaa atccaacaat atatttctcc atgaaggcct 600aacggtgaaa attggagatt ttggtttggc aacagtcaag tcgcgctgga gtggttctca 660gcaggttgaa caacctactg gctccatcct gtggatggcc ccagaggtga tccggatgca 720ggataacaac ccattcagct tccagtccga cgtctactcc tacggcattg tgctgtacga 780gctcatgacg ggggagctcc cttactccca catcaacaac cgtgatcaga tcatcttcat 840ggtgggccga ggctatgcct ccccagatct tagtaagctc tacaagaact gcccaaaagc 900aatgaagagg cttgtggccg actgtgtgaa gaaagttaag gaagaaaggc ctcttttnnc 960tcagatcctg tcttccattg agctgctcca acactctcta ccgaaaatca accggagtgc 1020ttctgagcca tccctgcacc gggcggccca cacggaggac atcaatgcct gcactctgac 1080cacatccccg agattgcccg tcttctagct gactctgcac ctgcgctcaa gccgctgtgg 1140gagaagtgaa gtcagcaggt accacctttc tgctcccttt ctgtggggac agagcttatc 1200ttcagagaag ctgctgctaa ggaccttcta gaccactcac agggccttaa cttcacgatg 1260ccttttctat ccaattctgg ccctgggaga aggaagccat tcgcgatgct ggtttgtcct 1320gctccccctc gaggtcccat gctcctgtgc tgagccttct ccagatgcac cagtggctgc 1380tgatggcatt atgggatctg gggccccagc tattgattgg ctaaatgagt aatttgagag 1440tcaagaaaaa aaagcactca gagggnnaaa aagtgactgg caggcaaacc agccatgaca 1500tgggggacat gttgatttcg ggaatcagct cctatgagca acacttatta cagaaagact 1560tctcttcaga gatgagatga gtggtgtggc gccaaacagg tagttttgca cataatgcac 1620caaacagccc aggactgtcg agactgtggc cgcctggagg agcctgcttt ggtactatgg 1680acttgacttt ggggacactt acctttcttg aaggtctcca gtgctttagg atggttttcc 1740acaagaggcg cttggcctgc ccctcccagt ctccaccctc tcagggagca gtcttccatt 1800gtgctaaatt agtcttccag gagctcgcct atggggcggg gccgctgggc cagccttgtc 1860tctacatcac atcatggtat gcaaggaagc cagaacacag gttttcttga taatttgggt 1920tttaattttg tttttattgc acctgcaaaa tacagttatc tgatgattct tcaattatgt 1980tattttaata aaataaatta aatgtaaaaa aaaaaaaa 2018 67 20 DNA artificialsequence antisense sequence 67 gttgctcata ggagctgatt 20 68 20 DNAartificial sequence antisense sequence 68 gtttggcgcc acaccactca 20 69 20DNA artificial sequence antisense sequence 69 caaggctggc ccagcggccc 2070 20 DNA artificial sequence antisense sequence 70 caggctcctccaggcggcca 20 71 20 DNA artificial sequence antisense sequence 71ccacaccact catctcatct 20 72 20 DNA artificial sequence antisensesequence 72 tcccgaaatc aacatgtccc 20 73 20 DNA artificial sequenceantisense sequence 73 cttccttctc ccagggccag 20 74 20 DNA artificialsequence antisense sequence 74 ctgacttcac ttctcccaca 20 75 20 DNAartificial sequence antisense sequence 75 gtcctccgtg tgggccgccc 20 76 20DNA artificial sequence antisense sequence 76 ccacaagcct cttcattgct 2077 20 DNA artificial sequence antisense sequence 77 catagcctcggcccaccatg 20 78 20 DNA artificial sequence antisense sequence 78cctgcatccg gatcacctct 20 79 20 DNA artificial sequence antisensesequence 79 cattccctga gccgtctgcc 20 80 20 DNA artificial sequenceantisense sequence 80 ctgctgccct cacaccactg 20 81 20 DNA artificialsequence antisense sequence 81 catctccatg ccacttgccc 20 82 20 DNAartificial sequence antisense sequence 82 ccggatcctg gcgcccgctc 20 83 20DNA artificial sequence antisense sequence 83 ccacaccaca catctcatct 2084 20 DNA artificial sequence antisense sequence 84 ccacaccacacttctcatct 20 85 20 DNA artificial sequence antisense sequence 85ccacacctca cttcacatct 20 86 20 DNA artificial sequence antisensesequence 86 ccactcctca cttcacaact 20 87 20 DNA artificial sequenceantisense sequence 87 cctctcctca cttcacaaca 20 88 20 DNA artificialsequence antisense sequence 88 ctctttctgt aataagtgtt 20 89 2510 DNA homosapiens 89 agcctcccgg ccccctcccc gcccgacagc ggccgctcgg gccccggctc 50tcggttataa gatggcggcg ctgagcggtg gcggtggtgg cggcgcggag 100 ccgggccaggctctgttcaa cggggacatg gagcccgagg ccggcgccgg 150 ccggcccgcg gcctcttcggctgcggaccc tgccattccg gaggaggtgt 200 ggaatatcaa acaaatgatt aagttgacacaggaacatat agaggcccta 250 ttggacaaat ttggtgggga gcataatcca ccatcaatatatctggaggc 300 ctatgaagaa tacaccagca agctagatgc actccaacaa agagaacaac350 agttattgga atctctgggg aacggaactg atttttctgt ttctagctct 400gcatcaatgg ataccgttac atcttcttcc tcttctagcc tttcagtgct 450 accttcatctctttcagttt ttcaaaatcc cacagatgtg gcacggagca 500 accccaagtc accacaaaaacctatcgtta gagtcttcct gcccaacaaa 550 cagaggacag tggtacctgc aaggtgtggagttacagtcc gagacagtct 600 aaagaaagca ctgatgatga gaggtctaat cccagagtgctgtgctgttt 650 acagaattca ggatggagag aagaaaccaa ttggttggga cactgatatt700 tcctggctta ctggagaaga attgcatgtg gaagtgttgg agaatgttcc 750acttacaaca cacaactttg tacgaaaaac gtttttcacc ttagcatttt 800 gtgacttttgtcgaaagctg cttttccagg gtttccgctg tcaaacatgt 850 ggttataaat ttcaccagcgttgtagtaca gaagttccac tgatgtgtgt 900 taattatgac caacttgatt tgctgtttgtctccaagttc tttgaacacc 950 acccaatacc acaggaagag gcgtccttag cagagactgccctaacatct 1000 ggatcatccc cttccgcacc cgcctcggac tctattgggc cccaaattct1050 caccagtccg tctccttcaa aatccattcc aattccacag cccttccgac 1100cagcagatga agatcatcga aatcaatttg ggcaacgaga ccgatcctca 1150 tcagctcccaatgtgcatat aaacacaata gaacctgtca atattgatga 1200 cttgattaga gaccaaggatttcgtggtga tggaggatca accacaggtt 1250 tgtctgctac cccccctgcc tcattacctggctcactaac taacgtgaaa 1300 gccttacaga aatctccagg acctcagcga gaaaggaagtcatcttcatc 1350 ctcagaagac aggaatcgaa tgaaaacact tggtagacgg gactcgagtg1400 atgattggga gattcctgat gggcagatta cagtgggaca aagaattgga 1450tctggatcat ttggaacagt ctacaaggga aagtggcatg gtgatgtggc 1500 agtgaaaatgttgaatgtga cagcacctac acctcagcag ttacaagcct 1550 tcaaaaatga agtaggagtactcaggaaaa cacgacatgt gaatatccta 1600 ctcttcatgg gctattccac aaagccacaactggctattg ttacccagtg 1650 gtgtgagggc tccagcttgt atcaccatct ccatatcattgagaccaaat 1700 ttgagatgat caaacttata gatattgcac gacagactgc acagggcatg1750 gattacttac acgccaagtc aatcatccac agagacctca agagtaataa 1800tatatttctt catgaagacc tcacagtaaa aataggtgat tttggtctag 1850 ctacagtgaaatctcgatgg agtgggtccc atcagtttga acagttgtct 1900 ggatccattt tgtggatggcaccagaagtc atcagaatgc aagataaaaa 1950 tccatacagc tttcagtcag atgtatatgcatttgggatt gttctgtatg 2000 aattgatgac tggacagtta ccttattcaa acatcaacaacagggaccag 2050 ataattttta tggtgggacg aggatacctg tctccagatc tcagtaaggt2100 acggagtaac tgtccaaaag ccatgaagag attaatggca gagtgcctca 2150aaaagaaaag agatgagaga ccactctttc cccaaattct cgcctctatt 2200 gagctgctggcccgctcatt gccaaaaatt caccgcagtg catcagaacc 2250 ctccttgaat cgggctggtttccaaacaga ggattttagt ctatatgctt 2300 gtgcttctcc aaaaacaccc atccaggcagggggatatgg tgcgtttcct 2350 gtccactgaa acaaatgagt gagagagttc aggagagtagcaacaaaagg 2400 aaaataaatg aacatatgtt tgcttatatg ttaaattgaa taaaatactc2450 tctttttttt taaggtggaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2500aaaaaaaccc 2510 90 20 DNA artificial sequence antisense sequence 90attttgaagg agacggactg 20 91 20 DNA artificial sequence antisensesequence 91 tggattttga aggagacgga 20 92 20 DNA artificial sequenceantisense sequence 92 cgttagttag tgagccaggt 20 93 20 DNA artificialsequence antisense sequence 93 atttctgtaa ggctttcacg 20 94 20 DNAartificial sequence antisense sequence 94 cccgtctacc aagtgttttc 20 95 20DNA artificial sequence antisense sequence 95 aatctcccaa tcatcactcg 2096 20 DNA artificial sequence antisense sequence 96 tgctgaggtgtaggtgctgt 20 97 20 DNA artificial sequence antisense sequence 97tgtaactgct gaggtgtagg 20 98 20 DNA artificial sequence antisensesequence 98 tgtcgtgttt tcctgagtac 20 99 20 DNA artificial sequenceantisense sequence 99 agttgtggct ttgtggaata 20 100 20 DNA artificialsequence antisense sequence 100 atggagatgg tgatacaagc 20 101 20 DNAartificial sequence antisense sequence 101 ggatgattga cttggcgtgt 20 10220 DNA artificial sequence antisense sequence 102 aggtctctgt ggatgattga20 103 20 DNA artificial sequence antisense sequence 103 attctgatgacttctggtgc 20 104 20 DNA artificial sequence antisense sequence 104gctgtatgga tttttatctt 20 105 20 DNA artificial sequence antisensesequence 105 tacagaacaa tcccaaatgc 20 106 20 DNA artificial sequenceantisense sequence 106 atcctcgtcc caccataaaa 20 107 20 DNA artificialsequence antisense sequence 107 ctctcatctc ttttcttttt 20 108 20 DNAartificial sequence antisense sequence 108 gtctctcatc tcttttcttt 20 10920 DNA artificial sequence antisense sequence 109 ccgattcaag gagggttctg20 110 20 DNA artificial sequence antisense sequence 110 tggatgggtgtttttggaga 20 111 20 DNA artificial sequence antisense sequence 111ctgcctggat gggtgttttt 20 112 20 DNA artificial sequence antisensesequence 112 ggacaggaaa cgcaccatat 20 113 20 DNA artificial sequenceantisense sequence 113 ctcatttgtt tcagtggaca 20 114 20 DNA artificialsequence antisense sequence 114 tctctcactc atttgtttca 20 115 20 DNAartificial sequence antisense sequence 115 actctctcac tcatttgttt 20 11620 DNA artificial sequence antisense sequence 116 gaactctctc actcatttgt20 117 20 DNA artificial sequence antisense sequence 117 tcctgaactctctcactcat 20 118 20 DNA artificial sequence antisense sequence 118ttgctactct cctgaactct 20 119 20 DNA artificial sequence antisensesequence 119 tttgttgcta ctctcctgag 20 120 20 DNA artificial sequenceantisense sequence 120 cttttgttgc tactctcctg 20 121 20 DNA artificialsequence antisense sequence 121 gctactctcc tgaactctct 20 122 20 DNAartificial sequence antisense sequence 122 ttccttttgt tgctactctc 20 12320 DNA artificial sequence antisense sequence 123 atttattttc cttttgttgc20 124 20 DNA artificial sequence antisense sequence 124 atatgttcatttattttcct 20 125 20 DNA artificial sequence antisense sequence 125tttattttcc ttttgttgct 20 126 20 DNA artificial sequence antisensesequence 126 tgttcattta ttttcctttt 20 127 20 DNA artificial sequenceantisense sequence 127 atttaacata taagcaaaca 20 128 20 DNA artificialsequence antisense sequence 128 ctgcctggta ccctgttttt 20 129 20 DNAartificial sequence antisense sequence 129 ctgcctggaa gggtgttttt 20 13020 DNA Artificial sequence Antisense sequence 130 ctgcctggta cggtgttttt20

What is claimed is:
 1. An oligonucleotide 8 to 50 nucleotides in lengthwhich is targeted to mRNA encoding human raf and which is capable ofinhibiting raf expression.
 2. The oligonucleotide of claim 1 which istargeted to mRNA encoding human A-raf.
 3. The oligonucleotide of claim 1which is targeted to mRNA encoding human B-raf.
 4. The oligonucleotideof claim 1 which is targeted to mRNA encoding human c-raf.
 5. Theoligonucleotide of claim 4 which is targeted to a translation initiationsite, 3′ untranslated region or 5′ untranslated region of mRNA encodinghuman c-raf.
 6. The oligonucleotide of claim 1 which has at least onephosphorothioate linkage.
 7. The oligonucleotide of claim 1 wherein atleast one of the nucleotide units of the oligonucleotide is modified atthe 2′ position of the sugar moiety.
 8. The oligonucleotide of claim 7wherein said modification at the 2′ position of the sugar moiety is a2′-O-alkyl, a 2′-O-alkyl-O-alkyl or a 2′-fluoro modification.
 9. Theoligonucleotide of claim 1 which is a chimeric oligonucleotide.
 10. Acomposition comprising the oligonucleotide of claim 1 and apharmaceutically acceptable carrier.
 11. The composition of claim 10further comprising a chemotherapeutic agent.
 12. A method of inhibitingthe expression of human raf in human cells or tissues which expresshuman raf comprising contacting said human cells or tissues with theoligonucleotide of claim
 1. 13. A method of treating or preventing acondition associated with the expression of human raf comprisingadministering to a human or cells thereof a therapeutically effectiveamount of the oligonucleotide of claim
 1. 14. The method of claim 13wherein said expression of human raf is abnormal expression.
 15. Themethod of claim 13 wherein said condition is a hyperproliferativecondition.
 16. The method of claim 15 wherein said hyperproliferativecondition is cancer.
 17. The method of claim 15 wherein saidhyperproliferative condition is angiogenesis or neovascularization. 18.The method of claim 17 wherein said angiogenesis or neovascularizationis ocular angiogenesis or neovascularization.
 19. The method of claim 16comprising administering the oligonucleotide in combination with achemotherapeutic agent.
 20. A method of inhibiting hyperproliferation ofcells comprising contacting hyperproliferating cells with theoligonucleotide of claim 1.